The species Stevia rebaudiana (“Stevia”) has been the subject of considerable research and development efforts directed at the purification of certain naturally occurring sweet glycosides of Stevia that have potential as non-caloric sweeteners. Sweet glycosides (also referred to as steviol glycosides) that may be extracted from Stevia include the six rebaudiosides (i.e., rebaudioside A to F), stevioside (the predominant glycoside in extracts from wild type Stevia), dulcosides, and sterebins.
Rebaudioside A (Reb A) is a sweet tasting glycoside component of Stevia, having roughly 250-450 times the sweetness of sucrose. Rebaudioside A is desirable for use in non-caloric sweeteners because of its favorable sweetness profile, regulatory approvals, customer acceptance, and minimal bitter aftertaste. Rebaudioside B (Reb B) and D (Reb D) also are sweet tasting glycoside components of Stevia that are of interest for their sweetness characteristics.
The natural extracts of Stevia as well as some processed versions thereof as well as synthetic counterparts typically include mixtures of glycosides. It has been desirable to purify these mixtures to obtain one or more of these glycosides in more pure form. For instance, a mixture might include, among other ingredients, a combination of Reb A, Reb, B, and Reb D. It has been desirable in some instances to treat these mixtures to recover a product that includes Reb A in more pure form while reducing the content of Reb B and/or D in the product. In other instances, it may be desirable to treat these mixtures to recover a product that includes Reb B and/or Reb D in more pure form while reducing the content of Reb A in the product. In still other instances, these mixtures are processed to recover a combination of products. For instance, if a mixture is treated to recover a mixture portion that is more pure with respect to Reb A with reduced Reb B and Reb D, another portion of the treated mixture generally can be recovered that has more pure Reb B and/or D and less Reb A content
Numerous methods have been reported for the purification of rebaudioside A from crude Stevia extracts containing rebaudioside A.
Japanese Publication No. 56121454 reports a method of separating stevioside and rebaudioside A at high purity and yield by crystallization. In the method a mixture of stevioside and rebaudioside A is extracted from the leaves and stalks of Stevia rebaudiana Bertoni by conventional process. The extract is dissolved in ≧70% aqueous solution of ethanol and rebaudioside A is selectively crystallized from the solution.
Japanese Patent 63173531 describes a method of extracting sweet glycosides from the Stevia rebaudiana plant. The first step of the process is to extract a liquid solution of sweet glycosides from the Stevia rebaudiana plant. Secondly, the liquid solution of sweet glycosides is passed through a non-polar porous resin and is eluted with a water-soluble organic solvent, preferably methanol. Thirdly, the eluted solution is concentrated and dried to give a powdery material. This procedure isolates a mixture of sweet glycosides, but does not isolate a single pure sweet glycoside such as rebaudioside A.
U.S. Patent Application Publication No. 2006/0083838 (Jackson et al.) reports a method of isolating and purifying rebaudioside A from commercially available Stevia rebaudiana starting material. The method comprises: (1) an ethanol (EtOH) formulation stage to formulate a selected EtOH solvent, (2) a first reflux stage using the Stevia starting material and optionally additional reflux stages using retentate isolated from a refluxed mixture or a stirred wash mixture, (3) optionally, one or more stirred wash stages, and (4) an ethanol purge and drying stage. In the reported method, an EtOH formulation stage is conducted in order to formulate a desired reflux solvent for use in the reflux step(s). Typically, the reflux solvent is a mixture of ethanol and water with about 5% to 15% by volume water. The reflux stage typically comprises providing a mixture of glycosides in the reflux solvent and refluxing the mixture for about 1 hour, cooling the mixture to improve the process yield, and filtering. The process further includes one or more energy-intensive refluxing steps that are typically conducted at a temperature of about 79° C. to 80° C. for about 1 hour. The stirred wash stage typically comprises providing a mixture of glycosides from a reflux stage and a solvent of pure ethanol, agitating the mixture at room temperature for about 15 minutes, and filtering. The method allegedly produces 100% pure, water-soluble rebaudioside A.
U.S. Pat. No. 5,962,678 (Payzant et al.) reports a method of extracting selected sweet glycosides from the Stevia rebaudiana plant. In the reported method, sweet glycosides are extracted from the Stevia plant and are processed to obtain individual components in a multi-step process. First, the Stevia plant is treated to extract an aqueous liquid solution containing mixed sweet glycosides. By using a series of ion exchange resins, the impure non-sweet glycosides are separated from the mixed sweet glycosides, which are dried. These dried mixed sweet glycosides, which still contain impurities, are then dissolved in a water-soluble organic solvent such as anhydrous methanol to form a solution. The solution is refluxed and is cooled to precipitate a first sweet glycoside component. This first sweet glycoside component, which is typically stevioside, can be recovered by filtration and may be further purified by the method described for the second component. The filtrate from the crystallization of the first precipitated sweet glycoside can be further treated to obtain a second sweet glycoside component by concentrating the filtrate by heating. Upon cooling the solution, a second sweet glycoside component precipitates which can be recovered. This second sweet glycoside component is typically rebaudioside A. It can be further purified by dissolving it in a water-soluble organic solvent such as methanol that may optionally contain a small amount of water. The solution is heated, refluxed, and finally cooled to precipitate the second sweet glycoside component at a higher purity. The precipitate can be recovered by filtration. This purification process can be repeated until a final crystallized solid of desired purity is obtained. The method reports Rebaudioside A purity levels of 90% or greater or 95% or greater.
U.S. Pat. No. 4,361,697 (Dobberstein et al.) reports a process for recovering diterpene glycosides from the Stevia rebaudiana plant. The process includes the steps of sequentially extracting plant material with a first solvent of intermediate polarity to extract plant substances which tend to interfere with a liquid chromatographic separation of the glycosides, and then with a second solvent of high polarity to extract glycosides, and chromatographically separating the extracted glycosides by introducing them onto a liquid chromatography column having a packing of an oxygen-containing organic stationary phase covalently bonded through a silicon atom to an inorganic support. The glycosides are eluted with a solvent of polarity that is higher than that of the first solvent but lower than that of the second solvent.
U.S. Pat. No. 4,892,938 (Giovanetto) reports a method for recovering steviosides from dried plant material of Stevia rebaudiana Bertoni by extraction and purification. An extract is obtained through treatment in water at a temperature from room temperature to about 65° C. with stirring and subsequent filtration and centrifugation. This extract is treated with calcium hydroxide, whereupon a precipitate is obtained by means of filtration or centrifugation. This precipitate is treated with a strongly acidic ion exchange resin and subsequently with a weakly basic ion exchange resin, filtered and dried.
U.S. Pat. No. 4,082,858 (DuBois) reports a method for the recovery of rebaudioside A from the leaves of Stevia rebaudiana plants. Final purification is achieved by liquid chromatography subsequently followed by an initial extraction with water and alkanol having from 1 to 3 carbon carbons, preferably methanol. It is also disclosed that water may be used as the initial solvent. Their preferred solvent at this stage is a liquid haloalkane having from 1 to 4 carbon atoms. The preferred second solvent is an alkanol having from 1 to 3 carbon atoms, while the preferred third solvent is an alkanol having from 1 to 4 carbon atoms and optionally minor amounts of water.
U.S. Patent Application No. 2006/0134292 (Abelyan et al.) reports a process for recovering sweet glycosides from Stevia rebaudiana plant material. The dried and powdered leaves are treated with water in the presence of a pectinase, cellulase, and alpha-amylase. The use of such enzymes is reported to considerably increase the extraction rate and facilitates the next stages of purification. The resulting extract is purified using treatment with calcium hydroxide and ultrafiltration. The permeate is passed through the column packed with bentonite and concentrated to syrup state under vacuum. The treatment with ethanol allows separating the practically pure rebaudioside A from the mixture. The rebaudioside A with high purity is obtained after washing the crystals with 88-95% of ethanol.
Other techniques include those reported, for example, in Japanese Publication Nos. 56121454; 56121455; 52062300; and 56121453 assigned to Ajinomoto Company, Inc, and in Chinese Publication No. 1243835 assigned to Hailin Stevia Rebaudium Sugar.
Due to their values as non-caloric sweeteners, improvements in the available methods for purifying glycosides such as Reb A, Reb B, and/or Reb D are desired. In particular, a method that allows for the separation of rebaudioside A from compositions containing rebaudioside B and/or rebaudioside D is highly desirable. This would allow recovery of a product that has more pure Reb A, a product that has more pure Reb B and/or D, or both kinds of products.